Axiocam503 monochrome

Samples were imaged using an upright microscope (Axio Imager A.2; Carl Zeiss, Oberkochen, Germany), with a 40× /1.3 and 100× /1.3 oil-immersion objective lens (EC Plan-Neofluar; Carl Zeiss), with transmitted light and reflected fluorescence observation using cooled charge-coupled device cameras (Axiocam 503 color and Axiocam 503 monochrome; Carl Zeiss). Cameras were equipped with sensors having a physical pixel length of 4.54 µm, yielding image pixel sizes of 113.5 nm/pixel (40× objective) or 45.4 nm/pixel (100× objective). Fluorescence was excited using a broadband light-emitting diode source (X-Cite 120 LED; Excelitas Technologies Corp., Waltham, MA), and a mercury lamp for brightfield illumination. Green fluorescence labelled samples were imaged using a GFP filter (Filter Set 09; Carl Zeiss) and red fluorescence labelled samples were imaged using a TxRed filter (Filter Set 00; Carl Zeiss). Images were acquired using a Zeiss software (ZEN 2Blue, Carl Zeiss, Carl Zeiss, https://www.zeiss.com).

Colony morphology was assessed from log phase cultures (OD₆₀₀ = 1) and streaked on Tryptic Soy agar. Plates were incubated 4 days at 37 °C and then imaged using a Zeiss microscope equipped with a Zeiss Plan Neo Fluor Z13/0.25 FWD objective. Images were taken with an Axiocam503 monochrome (Zeiss) camera and processed using ZEN 2 (blue edition). Sedimentation of bacterial aggregates was followed as reported earlier (24 (link)). Growth was initiated by inoculating mid-log phase cultures into fresh 7H9^OADC at an OD₆₀₀ of 0.05. Cultures were incubated at 37 °C with shaking and a sample of 1 ml of serially diluted culture was harvested every day and plated onto LB agar. After 4 days at 37 °C, colonies were counted to determine the colony forming unit (CFU).

To quantify bacterial loads, granulomas, cords and neutrophil recruitment, infected larvae were tricaine-anesthetized, positioned on 35-mm dishes, immobilized in 1% low-melting-point agarose and covered with water containing tricaine. Bright-field and fluorescence pictures of live infected embryos were taken with a Zeiss microscope equipped with a Zeiss Plan Neo Fluor Z 1x/0.25 FWD objective and a Axiocam503 monochrome (Zeiss) camera, with acquisition and processing using ZEN 2 (blue edition). Evaluation of intracellular mycobacterial growth, enumeration of macrophages recruitment, phagocytosis, mortality and granuloma organization, infected embryo were prepared for fixed microscopy. Animals were tricaine-anesthetized and fixed overnight at 4°C in 4% (vol/vol) paraformaldehyde in PBS, then washed twice in PBS, and transferred gradually from PBS to 50% (vol/vol) glycerol for microscopic observation. Confocal microscopy was performed using a Leica SPE upright microscope with 40x ACS APO 1.15 oil objective. Images were captured by LAS-AF software (Leica Microsystems).